ortho-anisidine This substance was considered by previous working groups, in 1981 (IARC, 1982) and 1987 (IARC, 1987). Since that time, new data have become available, and these have been incorporated into the monograph and taken into consideration in the present evaluation. 1. Exposure Data 1.1 Chemical and physical data 1.1.1 Nomenclature Chem. Abstr. Serv. Reg. No.: 90-04-0 Chem. Abstr. Name: 2-Methoxybenzenamine IUPAC Systematic Name: o-anisidine Synonyms: ortho-aminoanisole; 2-aminoanisole; ortho-aminomethoxybenzene; 2- aminomethoxybenzene; 1-amino-2-methoxybenzene; 2-methoxy-1-aminobenzene; ortho-methoxyaniline; 2-methoxyaniline; 2-methoxybenzenamine; ortho-methoxyphenylamine; 2-methoxyphenylamine 1.1.2 Structural and molecular formulae and relative molecular mass NH 2 OCH 3 C 7 H 9 NO Relative molecular mass: 123.15 1.1.3 Chemical and physical properties of the pure substance (a) Description: Yellowish liquid; becomes brownish on exposure to air (Budavari, 1996) (b) Boiling-point: 224 C (Lide, 1997) (c) Melting-point: 6.2 C (Lide, 1997) (d) Density: 1.096 g/cm 3 at 20 C (Lide, 1997) (e) Solubility: Slightly soluble in water; soluble in ethanol, diethyl ether and acetone (Lide, 1997) (f ) Stability: Susceptible to oxidation in air (National Toxicology Program, 1991) 49
50 IARC MONOGRAPHS VOLUME 73 (g) Octanol/water partition coefficient (P): log P, 0.95 (calculated), 1.18 (measured) (Verschueren, 1996). (h) Conversion factor: mg/m 3 = 5.04 ppm 1.2 Production and use Information available in 1995 indicated that ortho-anisidine was produced in Armenia, China, France, Germany, India, Japan, the Ukraine and the United Kingdom (Chemical Information Services, 1995). ortho-anisidine is used as a chemical intermediate (e.g. in the manufacture of azo or triphenylmethane dyes and pharmaceuticals), as a corrosion inhibitor for steel storage and as an antioxidant for some polymercaptan resins (National Toxicology Program, 1991). 1.3 Occurrence 1.3.1 Natural occurrence ortho-anisidine is not known to occur naturally. 1.3.2 Occupational exposure According to the 1981 83 National Occupational Exposure Survey (National Institute for Occupational Safety and Health, 1998), approximately 700 workers in the United States were potentially exposed to ortho-anisidine. Occupational exposure to orthoanisidine may occur during its production and during its use as a chemical intermediate, corrosion inhibitor or antioxidant. 1.3.3 Environmental occurrence ortho-anisidine has been identified in wastewater from chemical plants and from oil refineries, and in cigarette smoke (National Library of Medicine, 1998a). According to the United States Environmental Protection Agency Toxic Chemical Release Inventory for 1987, 1600 kg of ortho-anisidine were released into the air, 280 kg were discharged into water, and 110 kg were released onto the land from manufacturing and processing facilities in the United States. By 1996, 690 kg were released into the air and 13 kg discharged into water (National Library of Medicine, 1998b). 1.4 Regulations and guidelines The American Conference of Governmental Industrial Hygienists (1997) has recommended an 8-h time-weighted average threshold limit value of 0.5 mg/m 3, with a notation for potential dermal absorption, for occupational exposure to ortho-anisidine in workplace air. Similar values have been used as standards or guidelines in many other countries (International Labour Office, 1991). No international guidelines for ortho-anisidine in drinking-water have been established (WHO, 1993).
ortho-anisidine 51 2. Studies of Cancer in Humans 3. Studies of Cancer in Experimental Animals Previous evaluation ortho-anisidine hydrochloride was tested for carcinogenicity in one study in mice and one study in rats by oral administration in the diet. It produced transitional-cell carcinomas of the urinary bladder in animals of each species and sex (IARC, 1982). New studies In a model of urinary bladder carcinogenesis, groups of 15 male Fischer 344 rats, six weeks of age, were given 0.05% N-nitroso-N,4-hydroxybutylamine (NHBA) in the drinking-water for four weeks. They were then fed diets with or without a supplement of 1700 mg/kg diet (ppm) ortho-anisidine for the first two weeks and 425 ppm thereafter for an additional 30 weeks. Ten animals received ortho-anisidine without prior NHBA administration. The incidence of papillary or nodular hyperplasia in the urinary bladder, derived by assessing the number of lesions per unit length of mucosa, was significantly higher (p < 0.01) in the group receiving ortho-anisidine plus NHBA (13/16) than in the group given NHBA alone (2/13). No lesions were observed in animals receiving orthoanisidine alone (Ono et al., 1992). 4. Other Data Relevant to an Evaluation of Carcinogenicity and its Mechanisms 4.1 Absorption, distribution, metabolism and excretion 4.1.1 Humans 4.1.2 Experimental systems Horseradish peroxidase oxidized ortho-anisidine via a nitrogen-centred cation radical to the diimine, quinone imine and an azo dimer in vitro. The metabolism led to covalent binding to calf thymus DNA. Metabolites of ortho-anisidine were consistently more reactive with protein and glutathione than metabolites of para-anisidine (Thompson & Eling, 1991). [The Working Group noted that studies with mammalian enzymes have not been carried out.]
52 IARC MONOGRAPHS VOLUME 73 4.2 Toxic effects 4.2.1 Humans 4.2.2 Experimental systems The oral LD 50 of ortho-anisidine has been reported to be 2000 mg/kg bw in rats, 870 mg/kg bw in rabbits and 1400 mg/kg bw in mice. Its subacute effects include haematological changes, anaemia and nephrotoxicity (Prosolenko, 1975). ortho-anisidine induced methaemoglobinaemia in CBA mice and Alpk:APfSD rats after oral administration (Ashby et al., 1991). The authors suggested that their results indicate that ortho-anisidine is distributed and N-oxidized in rodents. Male and female B6C3F 1 mice were fed diets containing up to 30 000 mg/kg of diet (ppm) ortho-anisidine hydrochloride for seven weeks. A dose-dependent depression in mean body-weight gain of up to 40% was observed. The spleens of mice given doses > 10 000 mg/kg of diet were black and enlarged. Female mice that received doses of 2500 or 5000 mg/kg of diet for up to 103 weeks developed more cystic hyperplasia of the uterus and endometrium than did control animals. Mice of each sex at 30 000 mg/kg of diet had an increased incidence of hyperplasia of the bladder (National Cancer Institute, 1978). Feeding of diets containing up to 30 000 mg/kg (ppm) ortho-anisidine hydrochloride to Fischer 344 rats for seven weeks led to reductions in weight gain of up to 52% in males and 27% in females. Feeding of diets containing 1000 or 3000 mg/kg resulted in granular spleens in males but not in females; the spleens of rats of each sex given diets containing ortho-anisidine at > 10 000 mg/kg were dark and granular. A dose of 10 000 mg/kg of diet for up to 103 weeks resulted in depressions of body-weight gain of 21% in males and 11% in females. Male and female rats fed diets containing 5000 or 10 000 mg/kg orthoanisidine hydrochloride developed non-neoplastic lesions of the thyroid gland and kidney more frequently than did control animals (National Cancer Institute, 1978). 4.3 Reproductive and developmental effects 4.4 Genetic and related effects 4.4.1 Humans 4.4.2 Experimental systems (see Table 1 for references) ortho-anisidine did not induce reverse mutation in Escherichia coli or in Salmonella typhimurium strains TA100, TA1535, TA1537, TA1538, TA98 or YG1012. In the presence of exogenous metabolic activation, it induced mutations in strain YG1029, both YG strains having elevated levels of N-acetyltransferase. ortho-anisidine did not induce sex-linked recessive lethal mutations in Drosophila. It gave rise to gene mutations in
Table 1. Genetic and related effects of ortho-anisidine Test system Results a Dose b Reference (LED or HID) Without exogenous metabolic system With exogenous metabolic system Salmonella typhimurium TA100, TA98, TA1535, TA1537, reverse mutation 10 800 μg/plate Haworth et al. (1983) Salmonella typhimurium TA100, TA98, TA1535, TA1537, TA1538, 10 000 μg/plate Dunkel et al. (1985) reverse mutation Salmonella typhimurium YG1012 (TA1538 with N-acetyltransferase gene), Not reported Thompson et al. (1992) reverse mutation Salmonella typhimurium YG1029 (TA100 with N-acetyltransferase gene), + 62 μg/plate Thompson et al. (1992) reverse mutation Escherichia coli WP2uvrA, reverse mutation 10 000 μg/plate Dunkel et al. (1985) Drosophila melanogaster, sex-linked recessive lethal mutations 2000 ppm inj Yoon et al. (1985) DNA strand breaks/cross-links, mouse lymphoma L5178Y cells in vitro + 150 Garberg et al. (1988) Gene mutation, mouse lymphoma L5178Y cells, tk locus in vitro + + 123 Wangenheim & Bolcsfoldi (1988) Sister chromatid exchange, Chinese hamster ovary cells in vitro + + 38 Galloway et al. (1987) Chromosomal aberrations, Chinese hamster ovary cells in vitro + + 1200 Galloway et al. (1987) Cell transformation, Syrian hamster embryo cells, clonal assay + NT 500 Kerkaert et al. (1998) Host-mediated assay, Escherichia coli in blood of mouse, DNA repair in vivo 1300 po 1 Hellmer & Bolcsfoldi (1992) Host-mediated assay, Escherichia coli in blood of mouse, DNA repair in vivo + 310 ip 1 Hellmer & Bolcsfoldi (1992) DNA single-strand break, liver, thymus and testis of Sprague-Dawley rats 700 po 1 Ashby et al. (1991) in vivo DNA single-strand break, liver, kidney, spleen and bladder of Wistar rats 500 po 1 Ashby et al. (1991) in vivo DNA single-strand break, liver and bladder of Wistar rats in vivo 750 ip 1 Ashby et al. (1991) DNA breaks, bladder and colon of CD-1 mice in vivo + 690 po 1 Sasaki et al. (1998) ortho-anisidine 53
54 Table 1 (contd) Test system Results a Dose b Reference (LED or HID) Without exogenous metabolic system With exogenous metabolic system Unscheduled DNA synthesis, rat hepatocytes in vivo 1104 po 1 Ashby et al. (1991) Unscheduled DNA synthesis, rat kidney cells in vivo 500 ip 1 Tyson & Mirsalis (1985) Gene mutation, mouse bladder cells in vivo, laci transgenic model (+) 750 po 3 Ashby et al. (1994) Gene mutation, mouse liver cells in vivo, laci transgenic model - 750 po 10 Ashby et al. (1994) Micronucleus formation, CBA mouse bone-marrow cells in vivo 690 po 1 3 Ashby et al. (1991) Micronucleus formation, B6C3F 1 mouse bone-marrow cells in vivo 500 ip 3 Ashby et al. (1991) Micronucleus formation, mouse bone-marrow cells in vivo? 800 ip 1 Morita et al. (1997) Micronucleus formation, AP and Fischer 344 rat bone-marrow cells in vivo 1380 po 1 Ashby et al. (1991) Micronucleus formation, rats liver cells in vivo 1104 po 1 Ashby et al. (1991) Binding (covalent) to DNA, B6C3F 1 mouse bladder and liver in vivo 750 po 1 Ashby et al. (1994) Inhibition of gap-junctional intercellular communication, 3PC mouse keratinocytes in vitro + NT 1232 Jansen & Jongen (1996) IARC MONOGRAPHS VOLUME 73 a +, positive; (+), weakly positive;, negative; NT, not tested;?, inconclusive b LED, lowest effective dose; HID, highest ineffective dose; unless otherwise stated, in-vitro test, μg/ml; in-vivo test, mg/kg bw per day; inj, injection; po, oral; ip, intraperitoneal
ortho-anisidine 55 mouse lymphoma L5178Y cells in vitro both in the presence and absence of exogenous metabolic activation, while DNA breaks and cross-links were observed only in the presence of an exogenous metabolic system. Sister chromatid exchange and chromosomal aberrations were induced in Chinese hamster ovary cells in vitro, both in the presence and absence of exogenous metabolic activation. In the absence of exogenous metabolic activation, ortho-anisidine induced cell transformation in Syrian hamster embryo cells in vitro. ortho-anisidine did not bind covalently to DNA in mouse bladder or liver cells in vivo; it did not induce single-strand breaks in liver, thymus, testis, kidney, spleen or bladder DNA of rats treated in vivo, nor did it induce unscheduled DNA synthesis in rat hepatocytes or kidney cells in vivo. In mice treated in vivo, orthoanisidine gave rise to breaks in bladder and colon DNA but not in DNA of stomach, kidney, liver, lung, brain or bone marrow. ortho-anisidine induced DNA repair in E. coli in a host-mediated assay in male mice when the animals were treated intraperitoneally but not when they were treated by oral gavage. ortho-anisidine weakly induced gene mutation in the laci transgene in mouse bladder but not in liver. It did not induce micronuclei in bone marrow of mice or in bone-marrow or liver cells of rats treated in vivo. ortho-anisidine inhibited gap-junctional intercellular communication in mouse hepatocytes in the absence of an exogenous metabolic system in vitro. 5. Summary of Data Reported and Evaluation 5.1 Exposure data Exposure to ortho-anisidine may occur during its production and its use as a chemical intermediate, a corrosion inhibitor and an industrial antioxidant. 5.2 Human carcinogenicity data 5.3 Animal carcinogenicity data ortho-anisidine hydrochloride was tested for carcinogenicity in one study in mice and one study in rats by oral administration in the diet. It produced transitional-cell carcinomas of the urinary bladder in animals of each species and sex. 5.4 Other relevant data Limited information was available to the Working Group on the metabolism of ortho-anisidine. It was shown to be O-dealkylated in rat liver microsomes. ortho-anisidine at a high dose increased the incidence of hyperplasia of the bladder in male and female mice. No data were available on the developmental and reproductive effects of orthoanisidine.
56 IARC MONOGRAPHS VOLUME 73 No data were available on the genetic and related effects of ortho-anisidine in humans. No conclusion can be drawn about its genotoxicity in experimental animals in vivo; however, ortho-anisidine induced gene mutation in bladder cells in an assay in transgenic mice. There is no evidence that it has genotoxic effects in mammalian cells in vitro. ortho-anisidine was not mutagenic to bacteria. 5.5 Evaluation There is inadequate evidence in humans for the carcinogenicity of ortho-anisidine. There is sufficient evidence in experimental animals for the carcinogenicity of ortho-anisidine. Overall evaluation ortho-anisidine is possibly carcinogenic to humans (Group 2B). 6. References American Conference of Governmental Industrial Hygienists (1997) 1997 TLVs and BEIs, Cincinnati, OH, p. 16 Ashby, J., Lefevre, P.A., Tinwell, H., Brunborg, G., Schmezer, P., Pool-Zobel, B., Shanu-Wilson, R., Holme, J.A., Soderlund, E.J., Gulati, D. & Wojciechowski, J.P. (1991) The non-genotoxicity to rodents of the potent rodent bladder carcinogens o-anisidine and p-cresidine. Mutat. Res., 250, 115 133 Ashby, J., Short, J.M., Jones, N.J., Lefevre, P.A., Provost, G.S., Rogers, B.J., Martin, E.A., Parry, J.M., Burnette, K., Glickman, B.W. & H. Tinwell (1994) Mutagenicity of o-anisidine to the bladder of laci-transgenic B6C3F1 mice: Absence of 14 C or 32 P bladder DNA adduction. Carcinogenesis, 15, 2291 2296 Budavari, S., ed. (1996) The Merck Index, 12th Ed., Whitehouse Station, NJ, Merck & Co., Inc., p. 113 Chemical Information Services (1995) Directory of World Chemical Producers 1995/96 Edition, Dallas, TX, p. 66 Dunkel, V.C., Zeiger, E., Brusick, D., McCoy, E., McGregor, D., Mortelmans, K., Rosenkranz, H.S. & Simmon, V.F. (1985) Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ. Mutag., 7 (Suppl. 5), 1 248 Galloway, S.M., Armstrong, M.J., Reuben, C., Colman, S., Brown, B., Cannon, C., Bloom, A.D., Nakamura, F., Ahmed, M., Duk, S., Rimpo, J., Margolin, B.H., Resnick, M.A., Anderson, B. & Zeiger, E. (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. mol. Mutag., 10 (Suppl. 10), 1 175 Garberg, P., Akerblom, E.L. & Bolcsfoldi, G. (1988) Evaluation of a genotoxicity test measuring DNA-strand breaks in mouse lymphoma cells by alkaline unwinding and hydroxyapatite elution. Mutat. Res., 203, 155 176
ortho-anisidine 57 Haworth, S., Lawlor, T., Mortelmans, K., Speck, W. & Zeiger, E. (1983) Salmonella mutagenicity test results for 250 chemicals. Environ. Mutag., 5 (Suppl. 1), 1 142 Hellmer, L. & Bolcsfoldi, G. (1992) An evaluation of the E. coli K-12 uvrb/reca DNA repair hostmediated assay. II. In vivo results for 36 compounds tested in the mouse. Mutat. Res., 272, 161 173 IARC (1982) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 27, Some Aromatic Amines, Anthraquinones and Nitroso Compounds, and Inorganic Fluorides used in Drinking-water and Dental Preparations, Lyon, pp. 63 80 IARC (1987) IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Suppl. 7, Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs Volumes 1 to 42, Lyon, p. 57 International Labour Office (1991) Occupational Exposure Limits for Airborne Toxic Substances, 3rd. Ed. (Occupational Safety and Health Series No. 37), Geneva, pp. 26 27 Jansen, L.A.M. & Jongen, W.M.F. (1996) The use of initiated cells as a test system for the detection of gap junctional intracellular communication. Carcinogenesis, 17, 33 339 Kerckaert, G.A., LeBoeuf, R.A. & Isfort, R.J. (1998) Assessing the predictiveness of the Syrian hamster embryo cell transformation assay for determining the rodent carcinogenic potential of single ring aromatic/nitroaromatic amine compounds. Toxicol. Sci., 41, 189 197 Lide, D.R., ed. (1997) CRC Handbook of Chemistry and Physics, 78th Ed., Boca Raton, FL, CRC Press, p. 3-23 Morita, T., Asano, N., Awogi, T., Sasaki, Y.F., Sato, S., Shimada, H., Sutou, S., Suzuki, T., Wakata, A., Sofuni, T. & Hayashi, M. (1997) Evaluation of the rodent micronucleus assay in the screening of IARC carcinogens (Groups 1, 2A and 2B). The summary report of the 6th collaborative study by CSGMT/JEMS.MMS. Mutat. Res., 389, 3 122 National Cancer Institute (1978) Bioassay of o-anisidine Hydrochloride for Possible Carcinogenicity (Tech. Rep. Ser. No. 89; DHEW Publ. No (NIH) 78-1339), Washington DC, US Government Printing Office National Institute for Occupational Safety and Health (1998) National Occupational Exposure Survey 1981 83, Cincinnati, OH National Library of Medicine (1998a) Hazardous Substances Data Bank (HSDB), Bethesda, MD [Record No. 2073] National Library of Medicine (1998b) Toxic Chemical Release Inventory 1987 & 1996 (TRI87 & TRI96), Bethesda, MD National Toxicology Program (1991) NTP Chemical Repository Data Sheet: ortho-anisidine, Research Triangle Park, NC Ono, S., Kurata, Y, Shichino, Y., Sano, M. & Fukushima, S. (1992) Synergism of environmental carcinogens and promoters on bladder cancer development initiated by N-butyl-N-(4- hydroxybutyl)nitrosamine in F344 rats. Jpn. J. Cancer Res., 83, 955 963 Prosolenko, N.V. (1975) [Comparative toxicological evaluation of methoxyanilines (o- and p- anisidines)]. Tr. Khar k. Gos. med. Inst., 124, 11 14 (in Russian)
58 IARC MONOGRAPHS VOLUME 73 Sasaki, Y.F., Nishidate, E., Su, Y. Q., Matsusaka, N., Tsuda, S., Susa, N., Furukawa, Y. & Ueno, S. (1998) Organ-specific genotoxicity of the potent rodent bladder carcinogens o-anisidine and p-cresidine. Mutat. Res., 412, 155 160 Thompson, D.C. & Eling, T.E. (1991) Reactive intermediates formed during peroxidative oxidation of anisidine isomers. Chem. Res. Toxicol., 4, 474 481 Thompson, D.C., Josephy, P.D., Chu, J.W.K. & Eling, T.E. (1992) Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity. Mutat. Res., 279, 83 89 Tyson, C.K. & Mirsalis, J.C. (1985) Measurement of unscheduled DNA synthesis in rat kidney cells following in vivo treatment with genotoxic agents. Environ. Mutag., 7, 889 899 Verschueren, K. (1996) Handbook of Environmental Data on Organic Chemicals, 3rd Ed., New York, Van Nostrand Reinhold Co., pp. 205 206 Wangenheim, J. & Bolcsfoldi, G. (1988) Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds. Mutagenesis, 3, 193 205 WHO (1993) Guidelines for Drinking Water Quality, 2nd Ed., Vol. 1, Recommendations, Geneva Yoon, J.S., Mason, J.M., Valencia, R., Woodruff, R.C. & Zimmering, S. (1985) Chemical mutagenesis testing in Drosophila. IV. Results of 45 coded compounds tested for the National Toxicology Program. Environ. Mutag., 7, 349 367